ProjectReaching the effects of gastric bypass on diabetes and obesity without surgery

Researcher (PI)Jens Juul Holst

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsAdvanced Grant (AdG), LS4, ERC-2015-AdG

SummaryGastric bypass surgery results in massive weight loss and diabetes remission. The effect is superior to intensive medical treatment, showing that there are mechanisms within the body that can cure diabetes and obesity. Revealing the nature of these mechanisms could lead to new, cost-efficient, similarly effective, non-invasive treatments of these conditions. The hypothesis is that hyper-secretion of a number of gut hormones mediates the effect of surgery, as indicated by a series of our recent studies, demonstrating that hypersecretion of GLP-1, a hormone discovered in my laboratory and basis for the antidiabetic medication of millions of patients, is essential for the improved insulin secretion and glucose tolerance. But what are the mechanisms behind the up to 30-fold elevations in secretion of these hormones following surgery? Constantly with a translational scope, all elements involved in these responses will be addressed in this project, from detailed analysis of food items responsible for hormone secretion, to identification of the responsible regions of the gut, and to the molecular mechanisms leading to hypersecretion. Novel approaches for studies of human gut hormone secreting cells, including specific expression analysis, are combined with our advanced and unique isolated perfused gut preparations, the only tool that can provide physiologically relevant results with a translational potential regarding regulation of hormone secretion in the gut. This will lead to further groundbreaking experimental attempts to mimic and engage the identified mechanisms, creating similar hypersecretion and obtaining similar improvements as the operations in patients with obesity and diabetes. Based on our profound knowledge of gut hormone biology accumulated through decades of intensive and successful research and our successful elucidation of the antidiabetic actions of gastric bypass surgery, we are in a unique position to reach this ambitious goal.

Gastric bypass surgery results in massive weight loss and diabetes remission. The effect is superior to intensive medical treatment, showing that there are mechanisms within the body that can cure diabetes and obesity. Revealing the nature of these mechanisms could lead to new, cost-efficient, similarly effective, non-invasive treatments of these conditions. The hypothesis is that hyper-secretion of a number of gut hormones mediates the effect of surgery, as indicated by a series of our recent studies, demonstrating that hypersecretion of GLP-1, a hormone discovered in my laboratory and basis for the antidiabetic medication of millions of patients, is essential for the improved insulin secretion and glucose tolerance. But what are the mechanisms behind the up to 30-fold elevations in secretion of these hormones following surgery? Constantly with a translational scope, all elements involved in these responses will be addressed in this project, from detailed analysis of food items responsible for hormone secretion, to identification of the responsible regions of the gut, and to the molecular mechanisms leading to hypersecretion. Novel approaches for studies of human gut hormone secreting cells, including specific expression analysis, are combined with our advanced and unique isolated perfused gut preparations, the only tool that can provide physiologically relevant results with a translational potential regarding regulation of hormone secretion in the gut. This will lead to further groundbreaking experimental attempts to mimic and engage the identified mechanisms, creating similar hypersecretion and obtaining similar improvements as the operations in patients with obesity and diabetes. Based on our profound knowledge of gut hormone biology accumulated through decades of intensive and successful research and our successful elucidation of the antidiabetic actions of gastric bypass surgery, we are in a unique position to reach this ambitious goal.

Max ERC Funding

2 500 000 €

Duration

Start date: 2017-01-01, End date: 2021-12-31

Project acronymCFS modelling

ProjectChromosomal Common Fragile Sites: Unravelling their biological functions and the basis of their instability

Researcher (PI)Andres Joaquin Lopez-Contreras

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsStarting Grant (StG), LS4, ERC-2015-STG

SummaryCancer and other diseases are driven by genomic alterations initiated by DNA breaks. Within our genomes, some regions are particularly prone to breakage, and these are known as common fragile sites (CFSs). CFSs are present in every person and are frequently sites of oncogenic chromosomal rearrangements. Intriguingly, despite their fragility, many CFSs are well conserved through evolution, suggesting that these regions have important physiological functions that remain elusive. My previous background in genome editing, proteomics and replication-born DNA damage has given me the tools to propose an ambitious and comprehensive plan that tackles fundamental questions on the biology of CFSs. First, we will perform a systematic analysis of the function of CFSs. Most of the CFSs contain very large genes, which has made technically difficult to dissect whether the CFS role is due to the locus itself or to the encoded gene product. However, the emergence of the CRISPR/Cas9 technology now enables the study of CFSs on a more systematic basis. We will pioneer the engineering of mammalian models harbouring large deletions at CFS loci to investigate their physiological functions at the cellular and organism levels. For those CFSs that contain genes, the cDNAs will be re-introduced at a distal locus. Using this strategy, we will be able to achieve the first comprehensive characterization of CFS roles. Second, we will develop novel targeted approaches to interrogate the chromatin-bound proteome of CFSs and its dynamics during DNA replication. Finally, and given that CFS fragility is influenced both by cell cycle checkpoints and dNTP availability, we will use mouse models to study the impact of ATR/CHK1 pathway and dNTP levels on CFS instability and cancer. Taken together, I propose an ambitious, yet feasible, project to functionally annotate and characterise these poorly understood regions of the human genome, with important potential implications for improving human health.

Cancer and other diseases are driven by genomic alterations initiated by DNA breaks. Within our genomes, some regions are particularly prone to breakage, and these are known as common fragile sites (CFSs). CFSs are present in every person and are frequently sites of oncogenic chromosomal rearrangements. Intriguingly, despite their fragility, many CFSs are well conserved through evolution, suggesting that these regions have important physiological functions that remain elusive. My previous background in genome editing, proteomics and replication-born DNA damage has given me the tools to propose an ambitious and comprehensive plan that tackles fundamental questions on the biology of CFSs. First, we will perform a systematic analysis of the function of CFSs. Most of the CFSs contain very large genes, which has made technically difficult to dissect whether the CFS role is due to the locus itself or to the encoded gene product. However, the emergence of the CRISPR/Cas9 technology now enables the study of CFSs on a more systematic basis. We will pioneer the engineering of mammalian models harbouring large deletions at CFS loci to investigate their physiological functions at the cellular and organism levels. For those CFSs that contain genes, the cDNAs will be re-introduced at a distal locus. Using this strategy, we will be able to achieve the first comprehensive characterization of CFS roles. Second, we will develop novel targeted approaches to interrogate the chromatin-bound proteome of CFSs and its dynamics during DNA replication. Finally, and given that CFS fragility is influenced both by cell cycle checkpoints and dNTP availability, we will use mouse models to study the impact of ATR/CHK1 pathway and dNTP levels on CFS instability and cancer. Taken together, I propose an ambitious, yet feasible, project to functionally annotate and characterise these poorly understood regions of the human genome, with important potential implications for improving human health.

Max ERC Funding

1 499 711 €

Duration

Start date: 2016-05-01, End date: 2021-04-30

Project acronymEPIFISH

ProjectINNOVATIVE EPIGENETIC MARKERS FOR FISH DOMESTICATION

Researcher (PI)Jorge Manuel De Oliveira Fernandes

Host Institution (HI)NORD UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS9, ERC-2015-CoG

SummaryAquaculture is the fastest growing food production sector in the world, since there is an increasing demand for fish protein to feed a growing global population, which cannot be met by fisheries. In order to ensure the sustainability of this sector it is critical to domesticate and selectively improve the major commercial fish species. To date, the genetic markers used in selective breeding of fish account only for a fraction of the observed phenotypic variation. EPIFISH is a scientifically innovative and timely project that will address fish domestication and selection from a new perspective using a multidisciplinary approach. The rapid pace of substantial phenotypic changes during adaptation to new environmental conditions in fish undergoing domestication raises the original hypothesis that epigenetic mechanisms are involved in this process. Thus, the overarching aim of EPIFISH is to ascertain the importance of epigenetics in fish domestication using the Nile tilapia (Oreochromis niloticus) as model species. Specific objectives are i) to determine how selection affects the miRNA transcriptome and the epigenetic landscape during domestication, ii) to perform a functional characterization of miRNA variants and epigenetic alleles associated with growth, and iii) to validate them as potential epigenetic markers for future selective breeding programmes. The identification of epigenetic markers will be a ground-breaking element of EPIFISH with major impact on aquaculture biotechnology, since they will enable the development and application of epigenomic selection as a new feature in future selective breeding programmes. Moreover, the project outcomes will provide novel mechanistic insights into the role of epigenetics in fish domestication, which will surely open new horizons for future frontier research in epigenetics, namely transgenerational inheritance and nutritional epigenetics.

Aquaculture is the fastest growing food production sector in the world, since there is an increasing demand for fish protein to feed a growing global population, which cannot be met by fisheries. In order to ensure the sustainability of this sector it is critical to domesticate and selectively improve the major commercial fish species. To date, the genetic markers used in selective breeding of fish account only for a fraction of the observed phenotypic variation. EPIFISH is a scientifically innovative and timely project that will address fish domestication and selection from a new perspective using a multidisciplinary approach. The rapid pace of substantial phenotypic changes during adaptation to new environmental conditions in fish undergoing domestication raises the original hypothesis that epigenetic mechanisms are involved in this process. Thus, the overarching aim of EPIFISH is to ascertain the importance of epigenetics in fish domestication using the Nile tilapia (Oreochromis niloticus) as model species. Specific objectives are i) to determine how selection affects the miRNA transcriptome and the epigenetic landscape during domestication, ii) to perform a functional characterization of miRNA variants and epigenetic alleles associated with growth, and iii) to validate them as potential epigenetic markers for future selective breeding programmes. The identification of epigenetic markers will be a ground-breaking element of EPIFISH with major impact on aquaculture biotechnology, since they will enable the development and application of epigenomic selection as a new feature in future selective breeding programmes. Moreover, the project outcomes will provide novel mechanistic insights into the role of epigenetics in fish domestication, which will surely open new horizons for future frontier research in epigenetics, namely transgenerational inheritance and nutritional epigenetics.

Max ERC Funding

1 996 189 €

Duration

Start date: 2016-07-01, End date: 2021-06-30

Project acronymExtinction Genomics

ProjectExploring and exploiting the potential of extinct genome sequencing

Researcher (PI)Marcus Thomas Pius Gilbert

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS2, ERC-2015-CoG

SummaryPalaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

Palaeogenomics is the nascent discipline concerned with sequencing and analysis of genome-scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR-based Sanger sequencing, following the dawn of the second-generation sequencing revolution, it has rapidly become a reality. Indeed, so much so, that popular perception has moved away from if extinct species’ genomes can be sequenced, to when it will happen - and even, when will the first extinct animals be regenerated. Unfortunately this view is naïve, and does not account for the financial and technical challenges that face such attempts. I propose an exploration of exactly what the limits on genome reconstruction from extinct or otherwise historic/ancient material are. This will be achieved through new laboratory and bioinformatic developments aimed at decreasing the cost, while concomitantly increasing the quality of genome reconstruction from poor quality materials. In doing so I aim to build a scientifically-grounded framework against which the possibilities and limitations of extinct genome reconstruction can be assessed. Subsequently genomic information will be generated from a range of extinct and near-extinct avian and mammalian species, in order to showcase the potential of reconstructed genomes across research questions spanning at least three different streams of research: De-extinction, Evolutionary Genomics, and Conservation Genomics. Ultimately, achievement of these goals requires formation of a dedicated, closely knit team, focusing on both the methodological challenges as well as their bigger picture application to high-risk high-gain ventures. With ERC funding this can become a reality, and enable palaeogenomics to be pushed to the limits possible under modern technology.

Max ERC Funding

2 000 000 €

Duration

Start date: 2016-04-01, End date: 2021-03-31

Project acronymMATRICAN

ProjectMatrix during cancer progression

Researcher (PI)Janine Terra Erler

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS4, ERC-2015-CoG

SummaryThe extracellular matrix (ECM) is known to play a critical role in driving cancer progression, and yet we lack knowledge of its composition and structure. The goal of my ERC project is to investigate how alterations in biochemical composition and structural properties of the ECM during cancer progression impact on cell behaviour to drive metastasis, which is responsible for over 90% of cancer patient deaths. In order to do this, my lab has developed a method to in situ decellularise organs leaving structurally intact ECM scaffolds for subsequent analysis or for repopulation to study cell-ECM interactions in situ. We have deployed our method to decellularise primary tumour and metastatic organs in mice bearing orthotopic breast cancer tumours for subsequent quantitative global mass spectrometry (MS) proteomics, spatio-structural mapping of ECM components in 3D, and live imaging of repopulated cells. We observed fundamental alterations in ECM composition and structure between normal and tumour, and primary and metastatic tissue. We have selected two ECM components specifically upregulated in metastatic organs for subsequent validation. We discovered a marked decrease in proteins associated with fibrillogenesis in metastatic organs and will investigate the impact of this on metastatic ECM stiffness. We will decellularise organs from transgenic mouse models of breast and pancreatic cancer, at specific stages during cancer progression to determine the evolution of global ECM composition and structure, and how this impacts on cell behaviour through functional perturbation. Finally, we shall validate relevance of findings to human disease through use of human cancer lines and analysis of human patient samples. The research proposed will provide ground-breaking insight into how the ECM regulates cellular behaviour during normal and pathological conditions, and will test new strategies to combat metastasis that could be translated into the clinic to benefit cancer patients.

The extracellular matrix (ECM) is known to play a critical role in driving cancer progression, and yet we lack knowledge of its composition and structure. The goal of my ERC project is to investigate how alterations in biochemical composition and structural properties of the ECM during cancer progression impact on cell behaviour to drive metastasis, which is responsible for over 90% of cancer patient deaths. In order to do this, my lab has developed a method to in situ decellularise organs leaving structurally intact ECM scaffolds for subsequent analysis or for repopulation to study cell-ECM interactions in situ. We have deployed our method to decellularise primary tumour and metastatic organs in mice bearing orthotopic breast cancer tumours for subsequent quantitative global mass spectrometry (MS) proteomics, spatio-structural mapping of ECM components in 3D, and live imaging of repopulated cells. We observed fundamental alterations in ECM composition and structure between normal and tumour, and primary and metastatic tissue. We have selected two ECM components specifically upregulated in metastatic organs for subsequent validation. We discovered a marked decrease in proteins associated with fibrillogenesis in metastatic organs and will investigate the impact of this on metastatic ECM stiffness. We will decellularise organs from transgenic mouse models of breast and pancreatic cancer, at specific stages during cancer progression to determine the evolution of global ECM composition and structure, and how this impacts on cell behaviour through functional perturbation. Finally, we shall validate relevance of findings to human disease through use of human cancer lines and analysis of human patient samples. The research proposed will provide ground-breaking insight into how the ECM regulates cellular behaviour during normal and pathological conditions, and will test new strategies to combat metastasis that could be translated into the clinic to benefit cancer patients.

Max ERC Funding

1 997 500 €

Duration

Start date: 2016-09-01, End date: 2021-08-31

Project acronymnextDART

ProjectNext-generation Detection of Antigen Responsive T-cells

Researcher (PI)Sine Reker Hadrup

Host Institution (HI)DANMARKS TEKNISKE UNIVERSITET

Call DetailsStarting Grant (StG), LS6, ERC-2015-STG

SummaryOur current ability to map T-cell reactivity to certain molecular patterns poorly matches the huge diversity of T-cell recognition in humans. Our immune system holds approximately 107 different T-cell populations patrolling our body to fight intruding pathogens. Current state-of-the-art T-cell detection enables the detection of 45 different T-cell specificities in a given sample. Therefore comprehensive analysis of T-cell recognition against intruding pathogens, auto-immune attacked tissues or cancer is virtually impossible.
To gain insight into immune recognition and allow careful target selection for disease intervention, also on a personalized basis, we need technologies that allow detection of vast numbers of different T-cell specificities with high sensitivity in small biological samples.
I propose here a new technology based on multimerised peptide-major histocompatibility complex I (MHC I) reagents that allow detection of >1000 different T-cell specificities with high sensitivity in small biological samples. I will use this new technology to gain insight into the T-cell recognition of cancer cells and specifically assess the impact of mutation-derived neo-epitopes on T cell-mediated cancer cell recognition.
A major advantage of this new technology relates to the ability of coupling the antigen specificity to the T-cell receptor sequence. This will enable us to retrieve information about T-cell receptor sequences coupled with their molecular recognition pattern, and develop a predictor of binding between T-cell receptors and specific epitopes. It will ultimately enable us to predict immune recognition based on T-cell receptor sequences, and has the potential to truly transform our understanding of T cell immunology.
Advances in our understanding of T cell immunology are leading to massive advances in the treatment of cancer. The technologies I propose to develop and validate will greatly aid this process and have application for all immune related diseases.

Our current ability to map T-cell reactivity to certain molecular patterns poorly matches the huge diversity of T-cell recognition in humans. Our immune system holds approximately 107 different T-cell populations patrolling our body to fight intruding pathogens. Current state-of-the-art T-cell detection enables the detection of 45 different T-cell specificities in a given sample. Therefore comprehensive analysis of T-cell recognition against intruding pathogens, auto-immune attacked tissues or cancer is virtually impossible.
To gain insight into immune recognition and allow careful target selection for disease intervention, also on a personalized basis, we need technologies that allow detection of vast numbers of different T-cell specificities with high sensitivity in small biological samples.
I propose here a new technology based on multimerised peptide-major histocompatibility complex I (MHC I) reagents that allow detection of >1000 different T-cell specificities with high sensitivity in small biological samples. I will use this new technology to gain insight into the T-cell recognition of cancer cells and specifically assess the impact of mutation-derived neo-epitopes on T cell-mediated cancer cell recognition.
A major advantage of this new technology relates to the ability of coupling the antigen specificity to the T-cell receptor sequence. This will enable us to retrieve information about T-cell receptor sequences coupled with their molecular recognition pattern, and develop a predictor of binding between T-cell receptors and specific epitopes. It will ultimately enable us to predict immune recognition based on T-cell receptor sequences, and has the potential to truly transform our understanding of T cell immunology.
Advances in our understanding of T cell immunology are leading to massive advances in the treatment of cancer. The technologies I propose to develop and validate will greatly aid this process and have application for all immune related diseases.

Max ERC Funding

1 499 070 €

Duration

Start date: 2016-06-01, End date: 2021-05-31

Project acronymREPLICONSTRAINTS

ProjectDissecting the constraints that define the eukaryotic DNA replication program

Researcher (PI)Luis Ignacio Toledo Lazaro

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsStarting Grant (StG), LS3, ERC-2015-STG

SummaryDNA replication is essential for the perpetuation of life and, yet, it is also a major source of genomic instability that can lead to cancer and other human diseases. Despite the vast efforts invested in establishing the origins of genomic instability, the mechanisms that coordinate faithful genome duplication while ensuring its integrity remain unknown.
This dilemma is molecularly best exemplified by single stranded DNA (ssDNA), which inevitably results from unwinding the double helix due to replication fork progression, but is at the same time a vulnerable intermediate that can lead to severe genomic lesions. Thus, maintaining an appropriate balance of ssDNA is a paramount challenge for replicating cells. My own work has significantly contributed to this concept by showing that eukaryotic cells have limited resources to guard its ssDNA, and that exhaustion of these resources (due to increased overall levels of ssDNA) causes a lethal fragmentation of the genome termed ‘replication catastrophe’ (RC). To prevent this terminal scenario, ssDNA levels and DNA replication activity must be constrained by yet uncharacterized mechanisms. In eukaryotes, where DNA is simultaneously replicated at multiple sites throughout the genome, this represents a particularly challenging task. Understanding how this is molecularly accomplished could transform our view of the very principles of DNA replication regulation, and also reveal potential therapeutic avenues to exploit RC in the treatment for cancer.
With the present proposal I will address this challenge by investigating how ssDNA maintenance is enrooted in the regulatory principles of DNA replication. I will dissect the mechanisms that, globally and locally, constrain replication activity to prevent genomic instability. By using novel and innovative analytical tools, I aim to provide an unmatched picture of the DNA replication apparatus and to identify novel anticancer strategies based on provoking RC selectively in tumor cells.

DNA replication is essential for the perpetuation of life and, yet, it is also a major source of genomic instability that can lead to cancer and other human diseases. Despite the vast efforts invested in establishing the origins of genomic instability, the mechanisms that coordinate faithful genome duplication while ensuring its integrity remain unknown.
This dilemma is molecularly best exemplified by single stranded DNA (ssDNA), which inevitably results from unwinding the double helix due to replication fork progression, but is at the same time a vulnerable intermediate that can lead to severe genomic lesions. Thus, maintaining an appropriate balance of ssDNA is a paramount challenge for replicating cells. My own work has significantly contributed to this concept by showing that eukaryotic cells have limited resources to guard its ssDNA, and that exhaustion of these resources (due to increased overall levels of ssDNA) causes a lethal fragmentation of the genome termed ‘replication catastrophe’ (RC). To prevent this terminal scenario, ssDNA levels and DNA replication activity must be constrained by yet uncharacterized mechanisms. In eukaryotes, where DNA is simultaneously replicated at multiple sites throughout the genome, this represents a particularly challenging task. Understanding how this is molecularly accomplished could transform our view of the very principles of DNA replication regulation, and also reveal potential therapeutic avenues to exploit RC in the treatment for cancer.
With the present proposal I will address this challenge by investigating how ssDNA maintenance is enrooted in the regulatory principles of DNA replication. I will dissect the mechanisms that, globally and locally, constrain replication activity to prevent genomic instability. By using novel and innovative analytical tools, I aim to provide an unmatched picture of the DNA replication apparatus and to identify novel anticancer strategies based on provoking RC selectively in tumor cells.

Max ERC Funding

1 498 899 €

Duration

Start date: 2016-08-01, End date: 2021-07-31

Project acronymRNA ORIGAMI

ProjectRNA-protein Nanostructures for Synthetic Biology

Researcher (PI)Ebbe Sloth Andersen

Host Institution (HI)AARHUS UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS9, ERC-2015-CoG

SummarySynthetic biology aims at re-engineering organisms for practical applications by designing novel biomolecular components, networks, and pathways. The field is expected to lead to cheaper drugs, sustainable fuel production, efficient diagnosis and targeted therapies for diseases. However, a major obstacle to achieve these goals is our limited ability to rationally design biomolecular structure and function. By contrast, the field of DNA nanotechnology has so far demonstrated an unprecedented ability to design and self-assemble well-defined molecular shapes, although the production method of thermal annealing is not compatible with cells. We have recently demonstrated a breakthrough method, called RNA origami, which allows the design of RNA molecules that fold into well-defined nanoscale shapes during their synthesis by an RNA polymerase. In this proposal I aim at extending this technology to produce RNA-protein nanostructures and at demonstrating their application in synthetic biology. My primary scientific hypothesis is that understanding the folding process during synthesis will help us to design nanostructures that can be produced in cells. I will design a general RNA-protein architecture that is compatible with folding during synthesis. I will investigate folding kinetics to be able to design and program the dynamical folding process. Based on this, RNA-protein nanostructures will be designed, expressed in cells, and verified, for the formation of the desired shapes. We will develop new functionalities by both rational design and selection approaches with the aim of obtaining multivalent-binding and switching properties. Finally, the functional RNA-protein nanostructures will be applied in proof-of-concept experiments to demonstrate efficient, multivalent targeting of subcellular structures, biosensing of a variety of intracellular analytes, metabolic channeling of biosynthesis pathways, and complex control of transcriptional networks.

Synthetic biology aims at re-engineering organisms for practical applications by designing novel biomolecular components, networks, and pathways. The field is expected to lead to cheaper drugs, sustainable fuel production, efficient diagnosis and targeted therapies for diseases. However, a major obstacle to achieve these goals is our limited ability to rationally design biomolecular structure and function. By contrast, the field of DNA nanotechnology has so far demonstrated an unprecedented ability to design and self-assemble well-defined molecular shapes, although the production method of thermal annealing is not compatible with cells. We have recently demonstrated a breakthrough method, called RNA origami, which allows the design of RNA molecules that fold into well-defined nanoscale shapes during their synthesis by an RNA polymerase. In this proposal I aim at extending this technology to produce RNA-protein nanostructures and at demonstrating their application in synthetic biology. My primary scientific hypothesis is that understanding the folding process during synthesis will help us to design nanostructures that can be produced in cells. I will design a general RNA-protein architecture that is compatible with folding during synthesis. I will investigate folding kinetics to be able to design and program the dynamical folding process. Based on this, RNA-protein nanostructures will be designed, expressed in cells, and verified, for the formation of the desired shapes. We will develop new functionalities by both rational design and selection approaches with the aim of obtaining multivalent-binding and switching properties. Finally, the functional RNA-protein nanostructures will be applied in proof-of-concept experiments to demonstrate efficient, multivalent targeting of subcellular structures, biosensing of a variety of intracellular analytes, metabolic channeling of biosynthesis pathways, and complex control of transcriptional networks.

Max ERC Funding

1 999 935 €

Duration

Start date: 2016-04-01, End date: 2021-03-31

Project acronymSTC

ProjectSynaptic Tagging and Capture: From Synapses to Behavior

Researcher (PI)Sayyed Mohammad Sadegh Nabavi

Host Institution (HI)AARHUS UNIVERSITET

Call DetailsStarting Grant (StG), LS5, ERC-2015-STG

SummaryIt is shown that long-term potentiation (LTP) is the cellular basis of memory formation. However, since all but small fraction of memories are forgotten, LTP has been further divided into early LTP (e-LTP), the mechanism by which short-term memories are formed, and a more stable late LTP (L-LTP), by which long-term memories are formed. Remarkably, it has been shown that an e-LTP can be stabilized if it is preceded or followed by heterosynaptic L-LTP.
According to Synaptic Tagging and Capture (STC) hypothesis, e-LTP is stabilized by capturing proteins that are made by L-LTP induction. The model proposes that this mechanism underlies the formation of late associative memory, where the stability of a memory is not only defined by the stimuli that induce the change but also by events happening before and after these stimuli. As such, the model explicitly predicts that a short-term memory can be stabilized by inducing heterosynaptic L-LTP.
In this grant, I will put this hypothesis into test. Specifically, I will test two explicit predictions of STC model: 1) A naturally formed short-term memory can be stabilized by induction of heterosynaptic L-LTP. 2) This stabilization is caused by the protein synthesis feature of L-LTP. To do this, using optogenetics, I will engineer a short-term memory in auditory fear circuit, in which an animal transiently associates a foot shock to a tone. Subsequently, I will examine if optogenetic delivery of L-LTP to the visual inputs converging on the same population of neurons in the amygdala will stabilize the short-term tone fear memory.
To be able to engineer natural memory by manipulating synaptic plasticity I will develop two systems: 1) A two-color optical activation system which permits selective manipulation of distinct neuronal populations with precise temporal and spatial resolution; 2) An inducible and activity-dependent expression system by which those neurons that are activated by a natural stimulus will be optically tagged.

It is shown that long-term potentiation (LTP) is the cellular basis of memory formation. However, since all but small fraction of memories are forgotten, LTP has been further divided into early LTP (e-LTP), the mechanism by which short-term memories are formed, and a more stable late LTP (L-LTP), by which long-term memories are formed. Remarkably, it has been shown that an e-LTP can be stabilized if it is preceded or followed by heterosynaptic L-LTP.
According to Synaptic Tagging and Capture (STC) hypothesis, e-LTP is stabilized by capturing proteins that are made by L-LTP induction. The model proposes that this mechanism underlies the formation of late associative memory, where the stability of a memory is not only defined by the stimuli that induce the change but also by events happening before and after these stimuli. As such, the model explicitly predicts that a short-term memory can be stabilized by inducing heterosynaptic L-LTP.
In this grant, I will put this hypothesis into test. Specifically, I will test two explicit predictions of STC model: 1) A naturally formed short-term memory can be stabilized by induction of heterosynaptic L-LTP. 2) This stabilization is caused by the protein synthesis feature of L-LTP. To do this, using optogenetics, I will engineer a short-term memory in auditory fear circuit, in which an animal transiently associates a foot shock to a tone. Subsequently, I will examine if optogenetic delivery of L-LTP to the visual inputs converging on the same population of neurons in the amygdala will stabilize the short-term tone fear memory.
To be able to engineer natural memory by manipulating synaptic plasticity I will develop two systems: 1) A two-color optical activation system which permits selective manipulation of distinct neuronal populations with precise temporal and spatial resolution; 2) An inducible and activity-dependent expression system by which those neurons that are activated by a natural stimulus will be optically tagged.

Max ERC Funding

1 500 000 €

Duration

Start date: 2016-04-01, End date: 2021-03-31

Project acronymStemHealth

ProjectFoetal Intestinal Stem Cells in Biology and Health

Researcher (PI)Kim Bak Jensen

Host Institution (HI)KOBENHAVNS UNIVERSITET

Call DetailsConsolidator Grant (CoG), LS7, ERC-2015-CoG

SummaryThere is currently no medical cure for the millions of individuals affected by inflammatory bowel disease (IBD). These patients suffer from bleeding along the gastrointestinal tract due to epithelial ulceration, which causes severe abdominal pain, diarrhoea and malnutrition. This is due to the severely compromised integrity of the intestinal epithelium. I propose that patients with IBD will benefit from an intestinal epithelial transplant.
The objectives of this research programme are two fold. Firstly, I propose to perform preclinical testing of human intestinal epithelium to pave the way for their inclusion in clinical trials for IBD patients. This will be based on a combination of state-of-the-art cell culture methods with novel transplantation methodology. By combining analysis of intestinal epithelial cells from various developmental stages, I will be able to identify the most suitable source for transplantation and define how adult stem cells are specified in the tissue. Secondly, I will utilise an in vitro culture system to identify the transcriptional networks responsible for the maturation of the foetal intestinal epithelium. Tissue maturation currently constitutes a major roadblock in regenerative medicine as cells derived from foetal and pluripotent stem cells have foetal properties. Understanding this process will therefore improve our ability to generate sustainable sources of cells for transplantation, which is pivotal for future therapies relying on regenerative medicine and in vitro modelling of disease
The proposed research programme will have significant clinical and biological impact. Clinically, it provides the framework for initiating clinical trials for patients with IBD and protocols to obtain mature adult epithelium for in vitro disease modelling. From a biological perspective, we will gain insights into how specific signalling networks maintain specific cell states and dictate tissue maturation.

There is currently no medical cure for the millions of individuals affected by inflammatory bowel disease (IBD). These patients suffer from bleeding along the gastrointestinal tract due to epithelial ulceration, which causes severe abdominal pain, diarrhoea and malnutrition. This is due to the severely compromised integrity of the intestinal epithelium. I propose that patients with IBD will benefit from an intestinal epithelial transplant.
The objectives of this research programme are two fold. Firstly, I propose to perform preclinical testing of human intestinal epithelium to pave the way for their inclusion in clinical trials for IBD patients. This will be based on a combination of state-of-the-art cell culture methods with novel transplantation methodology. By combining analysis of intestinal epithelial cells from various developmental stages, I will be able to identify the most suitable source for transplantation and define how adult stem cells are specified in the tissue. Secondly, I will utilise an in vitro culture system to identify the transcriptional networks responsible for the maturation of the foetal intestinal epithelium. Tissue maturation currently constitutes a major roadblock in regenerative medicine as cells derived from foetal and pluripotent stem cells have foetal properties. Understanding this process will therefore improve our ability to generate sustainable sources of cells for transplantation, which is pivotal for future therapies relying on regenerative medicine and in vitro modelling of disease
The proposed research programme will have significant clinical and biological impact. Clinically, it provides the framework for initiating clinical trials for patients with IBD and protocols to obtain mature adult epithelium for in vitro disease modelling. From a biological perspective, we will gain insights into how specific signalling networks maintain specific cell states and dictate tissue maturation.